The aim of the proposed investigations is to elucidate the mechanism(s) for the synergistic interaction between spinally administered opioid and alpha-adrenergic agonists for the antinociceptive response. The use of opioids in the clinical management of pain is complicated by the unavoidable development of tolerance and physical dependence to these drugs. Development of a treatment regimen that would allow small doses of opioids in combination with non-opioid drugs to enhance the efficacy of opioids might allow more efficient pain management with less development of the undesirable opioid side effects. The antinociception observed for peripherally administered morphine may be a manifestation of the synergistic interaction that occurs between morphine administered concurrently at spinal and supraspinal sites. Supraspinally administered morphine activates a descending pain inhibitory control system which is mediated at the spinal level by released monoamines. Spinally administered morphine acts directly on opioid receptors in the spinal cord to produce antinociception. The spinal/supraspinal synergism may be a result of the synergistic interaction at the spinal level between the spinally administered opioid and the released norepinephrine; i.e., activation of opioid and non-opioid systems. Since both receptor types may be co-localized on the same spinal neurons and agonists of both receptors activate similar second messenger systems, this interaction may occur between the receptors, or between the G-proteins or second messengers activated by the receptors. Experiments will be performed to: (1) establish clearly the subtypes of the specific opioid and alpha adrenergic receptors involved in the antinociceptive synergistic interaction using receptor-selective agonists and antagonists. The ED50 values for the tail flick test in mice will be obtained and interactions between agonists will be evaluated isobolographically; (2) establish that any interaction which may occur between the two receptor types using competitive binding assays; (3) identify the G-proteins that interact with these receptors in spinal cord neuronal membranes using cholera toxin catalyzed ADP-ribosylation and [32p]NAD, or interaction with [32p]GTP azidoaniline; (4) examine the interaction between second messenger systems activated by opioid and alpha adrenergic agonists, specifically, inhibition of adenylyl cyclase activity and decreased intracellular calcium concentrations using spinal cord membranes, cultured dorsal root ganglia and clonal cells which express both opioid and alpha receptors. The decrease in spinal/supraspinal morphine synergism that occurs in morphine-tolerant mice has been proposed as a mechanism for development of tolerance to peripheral morphine. Thus, the proposed studies will also examine the changes in opioid/adrenergic interactions that may occur after chronic opioid treatment of either animals or cultured neuronal cells. These studies will provide insight into mechanisms of development of tolerance and physical dependence to morphine.